- Volume 162, Issue 6, 2016
Volume 162, Issue 6, 2016
- Cell Biology
-
-
-
ClpXP and ClpAP control the Escherichia coli division protein ZapC by proteolysis
More LessThe bacterial FtsZ-ring is an essential cytokinetic structure under tight spatiotemporal regulation. In Escherichia coli, FtsZ polymerization and assembly into the Z-ring is controlled on multiple levels through interactions with positive and negative regulators. Among these regulatory factors are ZapC, a Z-ring stabilizer, and the conserved protease ClpXP, which has been shown to degrade FtsZ protofilaments in preference to FtsZ monomers. Here we report that ZapC and ClpX interact in a protein–protein interaction assay, and that ZapC is degraded in a ClpXP-dependent manner in vivo. The SspB adaptor protein is not required for targeting ZapC to the ClpXP proteolytic machinery. A mutation disrupting the zapC ssrA-like sequence (zapCDD ) stabilizes ZapC consistent with a reduction in ClpXP-mediated ZapC degradation. ZapCDD retains the ability to interact with FtsZ and to promote bundling in vitro indicating that WT ZapC contains discrete FtsZ and ClpX recognition motifs. Additionally, ClpAP complexes are sufficient for degradation of ZapC in the absence of ClpX in vivo. Further, chromosomal expression of zapCDD suppresses filamentation of the temperature-sensitive ftsZ84 mutant, confirming the role of ZapC as a Z-ring stabilizer. Lastly, changes in ClpXP and ZapC levels lead to cell division effects, likely through their roles in modulating FtsZ assembly dynamics. Taken together, our results indicate that the Z-ring stabilizer ZapC is a substrate of both ClpXP and ClpAP in vivo. Our data also point to a more complex regulatory circuit that integrates FtsZ, ClpXP and ZapC in achieving Z-ring stability in E. coli and related species.
-
-
-
-
Conserved transmembrane glycine residues in the Shigella flexneri polysaccharide co-polymerase protein WzzB influence protein–protein interactions
More LessThe O antigen (Oag) component of lipopolysaccharides (LPS) is crucial for virulence and Oag chain-length regulation is controlled by the polysaccharide co-polymerase class 1 (PCP1) proteins. Crystal structure analyses indicate that structural conservation among PCP1 proteins is highly maintained, however the mechanism of Oag modal-chain-length control remains to be fully elucidated. Shigella flexneri PCP1 protein WzzBSF confers a modal-chain length of 10–17 Oag repeat units (RUs), whereas the Salmonella enterica Typhimurium PCP1 protein WzzBST confers a modal-chain length of ~16–28 Oag RUs. Both proteins share >70 % overall sequence identity and contain two transmembrane (TM1 and TM2) regions, whereby a conserved proline-glycine-rich motif overlapping the TM2 region is identical in both proteins. Conserved glycine residues within TM2 are functionally important, as glycine to alanine substitutions at positions 305 and 311 confer very short Oag modal-chain length (~2–6 Oag RUs). In this study, WzzBSF was co-expressed with WzzBST in S. flexneri and a single intermediate modal-chain length of ~11–21 Oag RUs was observed, suggesting the presence of Wzz:Wzz interactions. Interestingly, co-expression of WzzBSF with WzzBG305A/G311A conferred a bimodal LPS Oag chain length (despite over 99 % protein sequence identity), and we hypothesized that the proteins fail to interact. Co-purification assays detected His6-WzzBSF co-purifying with FLAG-tagged WzzBST but not with FLAG-tagged WzzBG305A/G311A, supporting our hypothesis. These data indicate that the conserved glycine residues in TM2 are involved in Wzz:Wzz interactions, and provide insight into key interactions that drive Oag modal length control.
-
- Environmental Biology
-
-
-
Functional roles of CymA and NapC in reduction of nitrate and nitrite by Shewanella putrefaciens W3-18-1
Shewanella putrefaciens W3-18-1 harbours two periplasmic nitrate reductase (Nap) gene clusters, NapC-associated nap-alpha (napEDABC) and CymA-dependent nap-beta (napDAGHB), for dissimilatory nitrate respiration. CymA is a member of the NapC/NirT quinol dehydrogenase family and acts as a hub to support different respiratory pathways, including those on iron [Fe(III)] and manganese [Mn(III, IV)] (hydr)oxide, nitrate, nitrite, fumarate and arsenate in Shewanella strains. However, in our analysis it was shown that another NapC/NirT family protein, NapC, was only involved in nitrate reduction, although both CymA and NapC can transfer quinol-derived electrons to a periplasmic terminal reductase or an electron acceptor. Furthermore, our results showed that NapC could only interact specifically with the Nap-alpha nitrate reductase while CymA could interact promiscuously with Nap-alpha, Nap-beta and the NrfA nitrite reductase for nitrate and nitrite reduction. To further explore the difference in specificity, site-directed mutagenesis on both CymA and NapC was conducted and the phenotypic changes in nitrate and nitrite reduction were tested. Our analyses demonstrated that the Lys-91 residue played a key role in nitrate reduction for quinol oxidation and the Asp-166 residue might influence the maturation of CymA. The Asp-97 residue might be one of the key factors that influence the interaction of CymA with the cytochromes NapB and NrfA.
-
-
- Genomics and systems biology
-
-
-
Antibiotic resistance due to an unusual ColE1-type replicon plasmid in Aeromonas salmonicida
Aeromonas salmonicida subsp. salmonicida is a fish pathogen known to have a rich plasmidome. In the present study, we discovered an isolate of this bacterium bearing an additional unidentified small plasmid. After having sequenced the DNA of that isolate by next-generation sequencing, it appeared that the new small plasmid is a ColE1-type replicon plasmid, named here pAsa7. This plasmid bears a functional chloramphenicol-acetyltransferase-encoding gene (cat-pAsa7) previously unknown in A. salmonicida and responsible for resistance to chloramphenicol. A comparison of pAsa7 with pAsa2, the only known ColE1-type replicon plasmid usually found in A. salmonicida subsp. salmonicida, revealed that even if both plasmids share a high structural similarity, it is still unclear if pAsa7 is a derivative of pAsa2 since they showed several mutations at the nucleotide level. Transcriptomic analysis revealed that the cat-pAsa4 gene, another chloramphenicol-acetyltransferase-encoding gene, found on the large plasmid pAsa4, was significantly more transcribed than cat-pAsa7. This was correlated with a higher chloramphenicol resistance for isolates bearing pAsa4 compared with the one having pAsa7. Finally, a phylogenetic analysis showed that both CAT-pAsa4 and CAT-pAsa7 proteins were in different clusters. The clustering was supported by the identity of residues involved in the catalytic site. In addition, to give a better understanding of the large drug-resistance panel of A. salmonicida, this study reinforces the hypothesis that A. salmonicida subsp. salmonicida is a considerable reservoir for mobile genetic elements such as plasmids.
-
-
- Host-microbe interaction
-
-
-
Glycolysis and pyrimidine biosynthesis are required for replication of adherent–invasive Escherichia coli in macrophages
Adherent–invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn’s disease (CD), a chronic inflammatory bowel condition. It has been proposed that AIEC-infected macrophages produce high levels of pro-inflammatory cytokines thus contributing to the inflammation observed in CD. AIEC can replicate in macrophages and we wanted to determine if bacterial replication was linked to the high level of cytokine production associated with AIEC-infected macrophages. Therefore, we undertook a genetic analysis of the metabolic requirements for AIEC replication in the macrophage and we show that AIEC replication in this niche is dependent on bacterial glycolysis. In addition, our analyses indicate that AIEC have access to a wide range of nutrients in the macrophage, although the levels of purines and pyrimidines do appear to be limiting. Finally, we show that the macrophage response to AIEC infection is indistinguishable from the response to the non-replicating glycolysis mutant (ΔpfkAB) and a non-pathogenic strain of E. coli, MG1655. Therefore, AIEC does not appear to subvert the normal macrophage response to E. coli during infection.
-
-
-
-
Elucidating population-wide mycobacterial replication dynamics at the single-cell level
More LessMycobacterium tuberculosis infections result in a spectrum of clinical outcomes, and frequently the infection persists in a latent, clinically asymptomatic state. The within-host bacterial population is likely to be heterogeneous, and it is thought that persistent mycobacteria arise from a small population of viable, but non-replicating (VBNR) cells. These are likely to be antibiotic tolerant and necessitate prolonged treatment. Little is known about these persistent mycobacteria, since they are very difficult to isolate. To address this, we have successfully developed a replication reporter system for use in M. tuberculosis. This approach, termed fluorescence dilution, exploits two fluorescent reporters; a constitutive reporter allows the tracking of bacteria, while an inducible reporter enables the measurement of bacterial replication. The application of fluorescence single-cell analysis to characterize intracellular M. tuberculosis identified a distinct subpopulation of non-growing mycobacteria in murine macrophages. The presence of VBNR and actively replicating mycobacteria was observed within the same macrophage after 48 h of infection. Furthermore, our results suggest that macrophage uptake resulted in enrichment of non- or slowly replicating bacteria (as revealed by d-cycloserine treatment); this population is likely to be highly enriched for persisters, based on its drug-tolerant phenotype. These results demonstrate the successful application of the novel dual fluorescence reporter system both in vitro and in macrophage infection models to provide a window into mycobacterial population heterogeneity.
-
-
-
Expanding the regulatory network that controls nitrogen fixation in Sinorhizobium meliloti: elucidating the role of the two-component system hFixL-FxkR
In Sinorhizobium meliloti, nitrogen fixation is regulated in response to oxygen concentration through the FixL-FixJ two-component system (TCS). Besides this conserved TCS, the field isolate SM11 also encodes the hFixL-FxkR TCS, which is responsible for the microoxic response in Rhizobium etli. Through genetic and physiological assays, we evaluated the role of the hFixL-FxkR TCS in S. meliloti SM11. Our results revealed that this regulatory system activates the expression of a fixKf orthologue (fixKa), in response to low oxygen concentration. Null mutations in either hFixL or FxkR promote upregulation of fixK1, a direct target of FixJ. Furthermore, the absence of this TCS translates into higher nitrogen fixation values as well as higher expression of fixN1 in nodules. Individual mutations in each of the fixK-like regulators encoded in the S. meliloti SM11 genome do not completely restrict fixN1 or fixN2 expression, pointing towards redundancy among these regulators. Both copies of fixN are necessary to achieve optimal levels of nitrogen fixation. This work provides evidence that the hFixL-FxkR TCS is activated in response to low oxygen concentration in S. meliloti SM11 and that it negatively regulates the expression of fixK1, fixN1 and nitrogen fixation.
-
- Physiology and metabolism
-
-
-
Heat shock response of thermophilic fungi: membrane lipids and soluble carbohydrates under elevated temperatures
The heat shock (HS) response is an adaptation of organisms to elevated temperature. It includes substantial changes in the composition of cellular membranes, proteins and soluble carbohydrates. To protect the cellular macromolecules, thermophilic organisms have evolved mechanisms of persistent thermotolerance. Many of those mechanisms are common for thermotolerance and the HS response. However, it remains unknown whether thermophilic species respond to HS by further elevating concentrations of protective components. We investigated the composition of the soluble cytosol carbohydrates and membrane lipids of the thermophilic fungi Rhizomucor tauricus and Myceliophthora thermophilaat optimum temperature conditions (41–43 °С), and under HS (51–53 °С). At optimum temperatures, the membrane lipid composition was characterized by a high proportion of phosphatidic acids (PA) (20–35 % of the total), which were the main components of the membrane lipids, together with phosphatidylcholines (PC), phosphatidylethanolamines (PE) and sterols (St). In response to HS, the proportion of PA and St increased, and the amount of PC and PE decreased. No decrease in the degree of fatty acid desaturation in the major phospholipids under HS was detected. The mycelium of all fungi at optimum temperatures contained high levels of trehalose (8–10 %, w/w; 60–95 % of the total carbohydrates), which is a hallmark of thermophilia. In contrast to mesophilic fungi, heat exposure decreased the trehalose level and the fungi did not acquire thermotolerance to lethal HS, indicating that trehalose plays a key role in this process. This pattern of changes appears to be conserved in the studied filamentous thermophilic fungi.
-
-
-
-
PhoB activation in non-limiting phosphate condition by the maintenance of high polyphosphate levels in the stationary phase inhibits biofilm formation in Escherichia coli
More LessPolyphosphate (polyP) degradation in Escherichia coli stationary phase triggers biofilm formation via the LuxS quorum sensing system. In media containing excess of phosphate (Pi), high polyP levels are maintained in the stationary phase with the consequent inhibition of biofilm formation. The transcriptional-response regulator PhoB, which is activated under Pi limitation, is involved in the inhibition of biofilm formation in several bacterial species. In the current study, we report, for the first time, we believe that E. coli PhoB can be activated in non-limiting Pi conditions, leading to inhibition of biofilm formation. In fact, PhoB was activated when high polyP levels were maintained in the stationary phase, whereas it remained inactive when the polymer was degraded or absent. PhoB activation was mediated by acetyl phosphate with the consequent repression of biofilm formation owing to the downregulation of c-di-GMP synthesis and the inhibition of autoinducer-2 production. These results allowed us to propose a model showing that PhoB is a component in the signal cascade regulating biofilm formation triggered by fluctuations of polyP levels in E. coli cells during stationary phase.
-
-
-
Trehalose is required for stress resistance and virulence of the Basidiomycota plant pathogen Ustilago maydis
Trehalose is an important disaccharide that can be found in bacteria, fungi, invertebrates and plants. In some Ascomycota fungal plant pathogens, the role of trehalose was recently studied and shown to be important for conferring protection against several environmental stresses and for virulence. In most of the fungi studied, two enzymes are involved in the synthesis of trehalose: trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2). To study the role of trehalose in virulence and stress response in the Basidiomycota maize pathogen Ustilago maydis, Δtps2 deletion mutants were constructed. These mutants did not produce trehalose as confirmed by HPLC analysis, showing that the single gene disruption impaired its biosynthesis. The mutants displayed increased sensitivity to oxidative, heat, acid, ionic and osmotic stresses as compared to the wild-type strains. Virulence of Δtps2 mutants to maize plants was extremely reduced compared to wild-type strains, possibly due to reduced capability to deal with the hostile host environment. The phenotypic traits displayed by Δtps2 strains were fully restored to wild-type levels when complemented with the endogenous UmTPS2 gene, or a chimeric construct having the Saccharomyces cerevisiae TPS2 ORF. This report demonstrates the presence of a single biosynthetic pathway for trehalose, and its importance for virulence in this model Basidiomycota plant pathogen.
-
-
-
Dissecting the role of histidine kinase and HOG1 mitogen-activated protein kinase signalling in stress tolerance and pathogenicity of Parastagonospora nodorum on wheat
More LessThe HOG1 mitogen-activated protein kinase (MAPK) pathway is activated through two-component histidine kinase (HK) signalling. This pathway was first characterized in the budding yeast Saccharomyces cerevisiae as a regulator of osmotolerance. The fungus Parastagonospora nodorum is the causal agent of septoria nodorum blotch of wheat. This pathogen uses host-specific effectors in tandem with general pathogenicity mechanisms to carry out its infection process. Genes showing strong sequence homology to S. cerevisiae HOG1 signalling pathway genes have been identified in the genome of P. nodorum. In this study, we examined the role of the pathway in the virulence of P. nodorum on wheat by disrupting putative pathway component genes: HOG1 (SNOG_13296) MAPK and NIK1 (SNOG_11631) hybrid HK. Mutants deleted in NIK1 and HOG1 were insensitive to dicarboximide and phenylpyrrole fungicides, but not a fungicide that targets ergosterol biosynthesis. Furthermore, both Δnik1 and Δhog1 mutants showed increased sensitivity to hyperosmotic stress. However, HOG1, but not NIK1, is required for tolerance to elevated temperatures. HOG1 deletion conferred increased tolerance to 6-methoxy-2-benzoxazolinone, a cereal phytoalexin. This suggests that the HOG1 signalling pathway is not exclusively associated with NIK1. Both Δnik1 and Δhog1 mutants retained the ability to infect and cause necrotic lesions on wheat. However, we observed that the Δhog1 mutation resulted in reduced production of pycnidia, asexual fruiting bodies that facilitate spore dispersal during late infection. Our study demonstrated the overlapping and distinct roles of a HOG1 MAPK and two-component HK signalling in P. nodorum growth and pathogenicity.
-
-
-
2-Deoxy-d-glucose is a potent inhibitor of biofilm growth in Escherichia coli
More LessEscherichia coli strain 15 (ATCC 9723), which forms robust biofilms, was grown under optimal biofilm conditions in NaCl-free Luria-Bertani broth (LB*) or in LB* supplemented with one of the non-metabolizable analogues 2-deoxy-d-glucose (2DG), methyl α-d-mannopyranoside (αMM), or methyl α-d-glucopyranoside (αMG). Biofilm growth was inhibited by mannose analogue 2DG even at very low concentration in unbuffered medium, and the maximal inhibition was enhanced in the presence of either 100 mM KPO4 or 100 mM MOPS, pH 7.5; in buffered medium, concentrations of 0.02 % (1.2 mM) or more inhibited growth nearly completely. In contrast, mannose analogue αMM, which should not be able to enter the cells but has been reported to inhibit biofilm growth by binding to FimH, did not exhibit strong inhibition even at concentrations up to 1.8 % (108 mM). The glucose analogue αMG inhibited biofilm growth, but much less strongly than did 2DG. None of the analogues inhibited planktonic growth or caused a change in pH of the unbuffered medium. Similar inhibitory effects of the analogues were observed in minimal medium. The effects were not strain-specific, as 2DG and αMG also inhibited the weak biofilm growth of E. coli K12.
-
- Regulation
-
-
-
CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia
SdhE is required for the flavinylation and activation of succinate dehydrogenase and fumarate reductase (FRD). In addition, SdhE is conserved in proteobacteria (α, β and γ) and eukaryotes. Although the function of this recently characterized family of proteins has been determined, almost nothing is known about how their genes are regulated. Here, the RsmA (CsrA) and RsmC (HexY) post-transcriptional and post-translational regulators have been identified and shown to repress sdhEygfX expression in Serratia sp. ATCC 39006. Conversely, the flagella master regulator complex, FlhDC, activated sdhEygfX transcription. To investigate the hierarchy of control, we developed a novel approach that utilized endogenous CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) genome-editing by a type I-F system to generate a chromosomal point mutation in flhC. Mutation of flhC alleviated the ability of RsmC to repress sdhEygfX expression, whereas RsmA acted in both an FlhDC-dependent and -independent manner to inhibit sdhEygfX. Mutation of rsmA or rsmC, or overexpression of FlhDC, led to increased prodigiosin, biosurfactant, swimming and swarming. Consistent with the modulation of sdhE by motility regulators, we have demonstrated that SdhE and FRD are required for maximal flagella-dependent swimming. Together, these results demonstrate that regulators of both metabolism and motility (RsmA, RsmC and FlhDC) control the transcription of the sdhEygfX operon.
-
-
Volumes and issues
-
Volume 170 (2024)
-
Volume 169 (2023)
-
Volume 168 (2022)
-
Volume 167 (2021)
-
Volume 166 (2020)
-
Volume 165 (2019)
-
Volume 164 (2018)
-
Volume 163 (2017)
-
Volume 162 (2016)
-
Volume 161 (2015)
-
Volume 160 (2014)
-
Volume 159 (2013)
-
Volume 158 (2012)
-
Volume 157 (2011)
-
Volume 156 (2010)
-
Volume 155 (2009)
-
Volume 154 (2008)
-
Volume 153 (2007)
-
Volume 152 (2006)
-
Volume 151 (2005)
-
Volume 150 (2004)
-
Volume 149 (2003)
-
Volume 148 (2002)
-
Volume 147 (2001)
-
Volume 146 (2000)
-
Volume 145 (1999)
-
Volume 144 (1998)
-
Volume 143 (1997)
-
Volume 142 (1996)
-
Volume 141 (1995)
-
Volume 140 (1994)
-
Volume 139 (1993)
-
Volume 138 (1992)
-
Volume 137 (1991)
-
Volume 136 (1990)
-
Volume 135 (1989)
-
Volume 134 (1988)
-
Volume 133 (1987)
-
Volume 132 (1986)
-
Volume 131 (1985)
-
Volume 130 (1984)
-
Volume 129 (1983)
-
Volume 128 (1982)
-
Volume 127 (1981)
-
Volume 126 (1981)
-
Volume 125 (1981)
-
Volume 124 (1981)
-
Volume 123 (1981)
-
Volume 122 (1981)
-
Volume 121 (1980)
-
Volume 120 (1980)
-
Volume 119 (1980)
-
Volume 118 (1980)
-
Volume 117 (1980)
-
Volume 116 (1980)
-
Volume 115 (1979)
-
Volume 114 (1979)
-
Volume 113 (1979)
-
Volume 112 (1979)
-
Volume 111 (1979)
-
Volume 110 (1979)
-
Volume 109 (1978)
-
Volume 108 (1978)
-
Volume 107 (1978)
-
Volume 106 (1978)
-
Volume 105 (1978)
-
Volume 104 (1978)
-
Volume 103 (1977)
-
Volume 102 (1977)
-
Volume 101 (1977)
-
Volume 100 (1977)
-
Volume 99 (1977)
-
Volume 98 (1977)
-
Volume 97 (1976)
-
Volume 96 (1976)
-
Volume 95 (1976)
-
Volume 94 (1976)
-
Volume 93 (1976)
-
Volume 92 (1976)
-
Volume 91 (1975)
-
Volume 90 (1975)
-
Volume 89 (1975)
-
Volume 88 (1975)
-
Volume 87 (1975)
-
Volume 86 (1975)
-
Volume 85 (1974)
-
Volume 84 (1974)
-
Volume 83 (1974)
-
Volume 82 (1974)
-
Volume 81 (1974)
-
Volume 80 (1974)
-
Volume 79 (1973)
-
Volume 78 (1973)
-
Volume 77 (1973)
-
Volume 76 (1973)
-
Volume 75 (1973)
-
Volume 74 (1973)
-
Volume 73 (1972)
-
Volume 72 (1972)
-
Volume 71 (1972)
-
Volume 70 (1972)
-
Volume 69 (1971)
-
Volume 68 (1971)
-
Volume 67 (1971)
-
Volume 66 (1971)
-
Volume 65 (1971)
-
Volume 64 (1970)
-
Volume 63 (1970)
-
Volume 62 (1970)
-
Volume 61 (1970)
-
Volume 60 (1970)
-
Volume 59 (1969)
-
Volume 58 (1969)
-
Volume 57 (1969)
-
Volume 56 (1969)
-
Volume 55 (1969)
-
Volume 54 (1968)
-
Volume 53 (1968)
-
Volume 52 (1968)
-
Volume 51 (1968)
-
Volume 50 (1968)
-
Volume 49 (1967)
-
Volume 48 (1967)
-
Volume 47 (1967)
-
Volume 46 (1967)
-
Volume 45 (1966)
-
Volume 44 (1966)
-
Volume 43 (1966)
-
Volume 42 (1966)
-
Volume 41 (1965)
-
Volume 40 (1965)
-
Volume 39 (1965)
-
Volume 38 (1965)
-
Volume 37 (1964)
-
Volume 36 (1964)
-
Volume 35 (1964)
-
Volume 34 (1964)
-
Volume 33 (1963)
-
Volume 32 (1963)
-
Volume 31 (1963)
-
Volume 30 (1963)
-
Volume 29 (1962)
-
Volume 28 (1962)
-
Volume 27 (1962)
-
Volume 26 (1961)
-
Volume 25 (1961)
-
Volume 24 (1961)
-
Volume 23 (1960)
-
Volume 22 (1960)
-
Volume 21 (1959)
-
Volume 20 (1959)
-
Volume 19 (1958)
-
Volume 18 (1958)
-
Volume 17 (1957)
-
Volume 16 (1957)
-
Volume 15 (1956)
-
Volume 14 (1956)
-
Volume 13 (1955)
-
Volume 12 (1955)
-
Volume 11 (1954)
-
Volume 10 (1954)
-
Volume 9 (1953)
-
Volume 8 (1953)
-
Volume 7 (1952)
-
Volume 6 (1952)
-
Volume 5 (1951)
-
Volume 4 (1950)
-
Volume 3 (1949)
-
Volume 2 (1948)
-
Volume 1 (1947)